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BR-112024027500-B1 - Polypeptide, nucleic acid, vector, host cell, method for producing the polypeptide, composition, use of the polypeptide, and method for promoting the proliferation of a cell in vitro.

BR112024027500B1BR 112024027500 B1BR112024027500 B1BR 112024027500B1BR-112024027500-B1

Abstract

POLYPEPTIDE, NUCLEIC ACID, VECTOR, HOST CELL, METHOD FOR PRODUCING THE POLYPEPTIDE, COMPOSITION, USE OF THE POLYPEPTIDE, AND METHOD FOR PROMOTING THE PROLIFERATION OF A CELL IN VITRO. This application provides a method for the biosynthesis of a structural material for the human body. A polypeptide has the amino acid sequence SEQ ID No. 4, 5, or 6. The recombinant humanized collagen type VII prepared in this application has high activity in promoting cell proliferation and does not produce an immune response when applied to the human body, and the method of preparation for this is novel and can obtain recombinant humanized collagen type VII on a large scale, being widely applied in the preparation of structural materials for the human body. The preparation method is applied to the fields of preparation of cutting-edge medical instruments, such as biological dressings, biomimetic materials of the human body, materials for plastic and aesthetic surgery, organoid culture materials, tissue injection and filling materials, skin repair and regeneration materials, oral mucosa repair and regeneration materials, cervical mucosa repair and regeneration materials, biological materials for gynecology and obstetrics, biological materials for artificial organs for 3D printing; cutting-edge cosmetic raw materials and cutting-edge medical auxiliary materials; (...).

Inventors

  • Xia Yang
  • Xiaobin Lan
  • Zhenrui He
  • Lingling Wang
  • Yongjian Zhang
  • Xin Liu
  • Haihong SONG

Assignees

  • Shanxi Jinbo Bio-Pharmaceutical Co., Ltd

Dates

Publication Date
20260317
Application Date
20230222
Priority Date
20220823

Claims (20)

  1. 1. Polypeptide, characterized by the fact that the amino acid sequence of the polypeptide is SEQ ID No. 4.
  2. 2. Polypeptide according to claim 1, characterized in that the polypeptide is a recombinant humanized type VII collagen.
  3. 3. Nucleic acid, characterized in that it comprises a nucleotide sequence encoding the polypeptide as defined in claim 1 or 2, wherein the nucleotide sequence consists of the nucleotide sequence of SEQ ID No. 7.
  4. 4. Nucleic acid according to claim 3, characterized in that it further comprises a nucleotide sequence encoding a purification tag.
  5. 5. Nucleic acid according to claim 4, characterized in that the purification label is a His label, a GST label, an MBP label, a SUMO label or a NusA label.
  6. 6. Nucleic acid according to any one of claims 3 to 5, characterized in that it further comprises a nucleotide sequence encoding a leader sequence.
  7. 7. Vector, characterized in that it comprises the nucleic acid as defined in any one of claims 3 to 6.
  8. 8. Vector according to claim 7, characterized in that it comprises an expression control element operably linked to nucleic acid.
  9. 9. Vector according to claim 8, characterized in that the expression control element is a promoter, a terminator, and/or an intensifier.
  10. 10. Host cell, characterized in that it comprises the nucleic acid as defined in any one of claims 3 to 6 or the vector as defined in any one of claims 7 to 9, wherein the host cell is a eukaryotic cell or a prokaryotic cell and the eukaryotic cell is a yeast cell.
  11. 11. Host cell according to claim 10, characterized in that the prokaryotic cell is an ESCHERICHIA COLI cell.
  12. 12. Host cell according to claim 11, characterized in that the Escherichia coli is E. coli BL21.
  13. 13. Method for producing the polypeptide as defined in claim 1 or 2, characterized in that it comprises: (1) cultivating the host cell as defined in any of claims 10 to 12 under an appropriate culture condition; (2) harvesting the host cells and/or the culture medium comprising the polypeptide; and (3) purifying the polypeptide.
  14. 14. Composition, characterized in that it comprises the polypeptide as defined in claim 1 or 2, the nucleic acid as defined in any one of claims 3 to 6, the vector as defined in any one of claims 7 to 9 and/or the host cell as defined in any one of claims 10 to 12.
  15. 15. Composition according to claim 14, characterized in that the composition is one or more of the following: biological dressings, biomimetic materials of the human body, materials for plastic and aesthetic surgery, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection and filling materials, ophthalmic materials, biological materials for gynecology and obstetrics, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, biomaterials for artificial organs for 3D printing, cosmetic raw materials, medicinal auxiliary materials and food additives.
  16. 16. Use of the polypeptide as defined in claim 1 or 2, characterized by being in the promotion of cell adhesion or promotion of cell proliferation IN VITRO or in the manufacture of a product or kit to promote cell adhesion or promote cell proliferation.
  17. 17. Use of the polypeptide as defined in claim 1 or 2, characterized by being in the manufacture of advanced medical devices, medicinal auxiliary materials, advanced cosmetic raw materials or food additives.
  18. 18. Use of the polypeptide as defined in claim 1 or 2, characterized by being used in the manufacture of biological dressings, biomimetic materials of the human body, materials for plastic and aesthetic surgery, organoid culture materials, cardiovascular stents, coatings, injection and tissue filling materials, ophthalmic materials, biological materials for gynecology and obstetrics, materials for nerve repair and regeneration, liver tissue materials and materials for vascular repair and regeneration, biological materials for artificial organs for 3D printing.
  19. 19. A method for promoting the proliferation of a cell in vitro, characterized in that it comprises bringing the cell into contact with the polypeptide as defined in claim 1 or 2.
  20. 20. Method according to claim 19, characterized in that the cell is an animal cell.

Description

[001] This application claims priority over Chinese Patent Application No. 202211017546.9 entitled “PREPARATION METHOD FOR BIOSYNTHESIS OF HUMAN BODY STRUCTURAL MATERIAL”, filed on August 23, 2022, which is incorporated herein by reference. Technical Field [002] This application pertains to the field of synthetic biotechnology and relates to a method of preparation for the biosynthesis of structural material for the human body. Foundation [003] The structural materials of the human body are primarily structural proteins, including collagen. These proteins have adhesion and support functions for cells and tissues and are the main component of the extracellular matrix. [004] Collagen is a type of protein widely distributed in human connective tissues and is also the most abundant protein in the human body, accounting for 25% to 35% of the total protein. Currently, it has been found that there are at least 28 subtypes of collagen in the human body, located in different tissues or organs. Among them, type VII collagen is a fibrous collagen distributed in the basement membrane area of stratified squamous epithelium, such as skin, buccal mucosa, and cervix. Thus, type VII collagen is also called basement membrane collagen. Type VII collagen is the main component of anchoring fibrils in the skin. These fibrils extend from the lamellar dendrites of the epidermal basement membrane to the dermal connective tissue, contributing to the adhesion between the epidermis and dermis. Animal experiments have shown that type VII collagen promotes fibroblast migration and cytokine secretion, as well as participating in skin damage and repair through tissue laminin; Type VII collagen also corrects epidermolysis bullosa of malnutrition through artificial injection of recombinant type VII collagen. [005] Currently, crude type VII collagen is obtained primarily by extraction from animal tissues or by lentiviral transfection. Unfortunately, type VII collagen has a relatively low content in the body, and the extraction process for type VII collagen is relatively complicated. Furthermore, animal-derived immune responses are a significant reason for limiting the application of collagen. In contrast, although lentiviral transfection is less immunogenic, it is difficult to operate and more limited in its ability to target genes. With the growing collagen industry in our country, the use of biosynthetic pathways for collagen production has become increasingly mature, especially in humanized collagen, which has been at the forefront globally. In 2021, the National Medical Products Administration conducted the naming and classification of biosynthetic collagen. Among them, recombinant humanized collagen refers to the total or partial amino acid sequence fragment encoded by the specific human collagen type gene prepared by recombinant DNA technology, or a combination containing functional fragments of human collagen. [006] Type VII collagen is a homotrimer composed of three identical α1 chains, and the molecular formula is α1α1α1(VII). The three α1(VII) chains twist together to form a triple-strand cordonal procollagen molecule. Procollagen molecules are secreted by cells, and excess protein fragments are removed from the ends by enzymatic treatment. After processing, the procollagen molecules are organized into long, thin bundles to form mature type VII collagen. Each α1 polypeptide chain contains a central collagen triple helix region, flanked by non-collagenous amino and carboxyl ends. The collagen structural region is a triple helix domain composed of a characteristic Gly-X-Y repeat sequence. Genetic mutation of type VII collagen causes synthesis disorders or structural abnormalities of collagen, leading to varying degrees of blistering. Symptoms involve the skin, oral mucosa, esophagus, and other sites. Although most are non-directed mutations, missense mutations can still occur, resulting in collagen retention and impaired folding in cells, affecting the connection between the vaginal mucosa and the lamina propria of the basement membrane. [007] Currently, type VII collagen has been obtained mainly through enzymatic digestion and lentiviral transfection. Enzymatic digestion refers to the extraction of type VII collagen derivatives by treating animal-derived tissues with proteases. However, collagen extracted by this method has lost its original biological activity and cannot perform the true function of collagen. Lentiviral transfection refers to the construction of a retroviral vector, followed by cell transfection and, finally, the purification of type VII collagen. However, the preparation process of this method has flaws, as preparation is difficult and it is not easy to obtain high-purity viruses; the integration of lentiviral vectors into the host genome is random, which can interfere with gene expression at the insertion site and neighboring genes; it is difficult to obtain precise control of the number of integrated copies; and